1. Field of the Invention
The present invention relates to optical indicator compounds, and the methods for their preparation and use, which provide optical signals upon contact with oxidants, particularly hydrogen peroxide. In particular, the invention relates to fluorogenic indicators for hydrogen peroxide and their use in analytical systems, such as diagnostic test systems, which are based on the generation and detection of hydrogen peroxide in response to the analyte under determination.
2. Description of the Prior Art
Many analytical systems involve the measurement of an oxidative substance as the ultimately detected substance. The analyte under determination may itself be such oxidant or the analyte may participate in a chemical, biological, immunological, or the like reaction that produces or destroys a measurable oxidant. These oxidants include substances such as hydrogen peroxide, ozone, periodates, peracids, and superoxides.
In particular, the determination of oxidative enzyme activity is important in analytical chemistry and biochemistry because of its usefulness in clinical diagnostic systems. Among the more commonly studied oxidative enzymes are the oxidases which produce hydrogen peroxide, such as glucose oxidase, xanthine oxidase, and cholesterol oxidase. The hydrogen peroxide generated by the action of such enzymes on their substrates is generally quantitated by oxidation-reduction reactions with various types of optical indicators, usually in the presence of peroxidase. One of the most sensitive means for quantitating hydrogen peroxide is by the use of fluorogenic peroxidase substrates which yield fluorescent products upon peroxidase-catalyzed reaction with hydrogen peroxide.
The literature contains relatively few examples of fluorogenic peroxidase substrates. Some such indicators that have been reported in the literature are homovanillic acid, 3-(p-hydroxyphenyl)propionic acid, 4-amino-1-1,5-benzodiazepine-3-carbonitrile, and diacetyldichlorofluorescin [K. Zaitsu and Y. Ohkura, Anal. Biochem. 109:109(1980), H. Corrodi and B. Weidinius, Acta Chem. Scand. 19:1854(1965), Y. Okamoto et al, Chem. Pharm. Bull. 28:2325(1980), A. S. Keston and R. Brandt, Anal. Biochem. 11:1(1965), and U.S. Pat. No. 4,269,938]. The fluorometric determination of hydrogen peroxide has also been accomplished using the fluorescent compound 6-methoxy-7-hydroxy-1,2-benzopyrone which is oxidized to a nonfluorescent product [W. A. Andreae, Nature 175:859(1955)].
The known fluorogenic peroxidase substrates suffer from several disadvantages particularly as applied to analytical systems for determining analytes in proteinaceous reaction mixtures, e.g., in immunoassays. Such disadvantages include lack of sensitivity to hydrogen peroxide, instability in aqueous media, excitation wavelengths for the fluorescent product within the range of excitation wavelengths for proteins present in the reaction mixture, and an affinity for binding to proteins present in the reaction mixture.